Choleragen activates adenylate cyclase by catalyzing, in the presence of NAD, the ADP-ribosylation of Gs alpha, the stimulatory GTP-binding protein of the cyclase system. Kahn and Gilman (J. Biol. Chem. 261, 7906-7911 (1986)) identified another GTP-binding protein termed ADP-ribosylation factor (ARF) that stimulated this reaction. It was proposed that the toxin substrate is an ARF-Gs alpha complex and that ARF may have a physiological role in regulation of Gs alpha activity. ARF, purified from bovine brain membranes, enhanced not only the ADP-ribosylation of Gs alpha, but also Gs alpha-independent choleragencatalyzed reactions. These are the (1) ADP- ribosylation of agmatine, a low molecular weight guanidino compound; (2) ADP-ribosylation of several of several proteins unrelated to Gs alpha; and (3) auto-ADP-ribosylation of the toxin A1 peptide. These reactions, as well as the ADP-ribosylation of ARF itself, were stimulated by GTP or stable GTP analogues such as guanyl-5'-yl imido-beta gamma-diphosphate and guanosine 5'-0- )+(3-thio-triphosphate); GDP and guanosine-5'-0-(2- thiodiphosphate) were inactive. These observations are consistent with the conclusion that ARF interacts directly with the A subunit of choleragen in a GTP-dependent fashion thereby enhancing catalytic activity manifest as transfer of ADP-ribose to Gs alpha and other proteins, to the toxin A1 peptide, or to agmatine. We have also purified two other soluble factors that enhanced the ability of choleragen to ADP-ribosylate Gs alpha. Each exhibited properties similar to ARF leading to the speculation that a family of ARF-like proteins may exist in animal cells.